SMAD-activation by recombinant GDF15 in myeloma cell lines.

<p>A. Phosphorylation of SMAD1/5 or SMAD2 was determined using immunoblotting in IH-1 cells treated with BMP-9 (0.5 ng/mL), activin A (25 ng/mL) or indicated concentrations of GDF15 (100–400 ng/mL) for 1 hour. B. INA-6 cells were treated with GDF15 (200 ng/mL) and the inhibitor SB431542 (0–2.5 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. C. INA-6 cells were transiently transfected with siRNAs targeting <i>ACVR1B/ALK4</i>, <i>ACVR1C/ALK7</i>, <i>TGFBR1/ALK5</i> and a non-targeting control siRNA. Two days after transfection the cells were treated with GDF15 (200 ng/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. D. Knock-down of receptors by siRNA in cells used in (C) as shown by QRT-PCR. Gene expression was calculated with the comparative ΔCt-method with <i>GAPDH</i> as housekeeping gene. The error bars indicate SEM of three independent experiments. Asterisks above bars indicate the degree of significance for downregulation of each gene compared to control (*, P≤0.05; **, P≤0.01; and ***, P≤0.001). E. INA-6 cells were treated with GDF15 (100 ng/mL) and a neutralizing TGFBR2 antibody (10–15 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. F. INA-6 cells were treated with GDF15 (100 ng/mL) and the indicated soluble receptors (5 μg/mL for all except endoglin, which was 1 μg/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. Antibody staining towards GAPDH was used as loading control for all Western blots. The experiments were performed 2–3 times each. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081.</p

Activation of SMAD2 by recombinant GDF15 was caused by TGF-β.

<p><i>In vitro</i> differentiated macrophages (A) or THP-1 cells (B) were treated with increasing doses of recombinant GDF15 (R&D Systems, Lot# EHF0914051) or TGF-β for four hours. C. INA-6 cells were treated with increasing doses of TGF-β for 1 hour. D. INA-6 cells were treated for 1 hour with the indicated doses of TGF-β, GDF15 (Abcam) or GDF15 (Peprotech). E. INA-6 cells were treated for 1 hour with GDF15 (Peprotech) or TGF-β pre-treated with neutralizing antibodies targeting GDF15 or TGF-β. For C-E, the experiments were performed in RPMI with 0.1% bovine serum albumin (BSA). Phosphorylation of SMAD2 was determined using immunoblotting and GAPDH, ERK1/2 or SMAD2/3 antibodies were used as loading controls. All experiments were performed at least three times, except for D and E, which were performed twice.</p

SMAD-activation by recombinant GDF15 in THP-1-cells and <i>in vitro</i> differentiated macrophages.

<p>A. Monocytic THP-1 cells were treated with GDF15 (50, 100 or 200 ng/mL), BMP-9 (50 ng/mL) or activin A (100 ng/mL) for 4 hours. B. THP-1 cells were treated with GDF15 (100 ng/mL) for various time-points. C. <i>In vitro</i> differentiated macrophages were treated with indicated soluble receptors in the presence of TGF-β (1 ng/mL) or GDF15 (200 ng/mL) for four hours. Phosphorylation of SMAD2 was determined using immunoblotting and GAPDH was used as loading control for all Western blots. Each experiment was performed once. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081.</p